Conclusions: Ki-67 overexpression is associated with features of aggressive bladder TCC and adds independent prognostic information to standard pathologic features for prediction of clinical outcome after radical cystectomy.

Ki-67

MIB-1

p53

transitional cell carcinoma

biomarkers

Radical cystectomy remains the most common treatment for patients with muscle-invasive bladder cancer or for patients with nonmuscle-invasive disease that is refractory to transurethral resection (TUR) with intravesical therapy. Despite advances in surgical technique and improved understanding of the role of pelvic lymphadenectomy, 5-year disease-specific survival remains 50% to 60% (1, 2). Moreover, while providing important prognostic information in transitional cell carcinoma (TCC) of the urinary bladder, current clinical and pathologic variables have a limited ability to predict tumor recurrence, progression, or patient survival. With availability of effective systemic chemotherapy, there is a clear need for accurate predictors of failure after local therapy with curative intent. Biomarkers may be helpful for selecting patients best suited for early systemic intervention and for sparing patients who have undergone cystectomy from the morbidity associated with local adjuvant or salvage radiation therapy.

Cell proliferation is a hallmark of cancer. Ki-67 is a nuclear protein expressed by proliferating cells and can be observed immunohistochemically. Nuclear Ki-67 antigen expression is a measure of cell growth fraction and hence biological aggressiveness of a malignancy (3). This marker has shown promise as an independent prognosticator of patient outcome in several malignancies (4–6). Most recently, investigations established Ki-67 antigen as an independent predictor of recurrence, progression, and response to immunotherapy in patients with nonmuscle-invasive TCC (7–10). To date, few studies explored the predictive role of Ki-67 after radical cystectomy for advanced TCC (11, 12). These studies were flawed due to small size, and they did not do subgroup analyses to determine the significance of Ki-67 for different patient groups.

We determined the association of Ki-67 expression with clinicopathologic characteristics and oncologic outcomes in patients with TCC treated with radical cystectomy.

Materials and Methods

Patient population. All studies were undertaken with the approval and institutional oversight of the Institutional Review Board for the Protection of Human Subjects at the University of Texas Southwestern Medical School. The study comprised 226 patients who underwent radical cystectomy with bilateral lymphadenectomy. The indications for radical cystectomy were tumor invasion into the muscularis propria or prostatic stroma or Ta, T1, or carcinoma in situ refractory to TUR with intravesical chemotherapy and/or immunotherapy. No patient had distant metastatic disease at the time of cystectomy. Malignant lymph nodes from 50 of the 61 patients who had metastases to lymph nodes were available for assessment. The clinical stage was assigned by the operative surgeon according to the 1997 tumor-node-metastasis system. For each patient, comprehensive clinical and pathologic data elements were collected and entered into an institutional review board–approved database. Multiple data reviews and quality checks were done to assure the accuracy and completeness of data elements. Chemotherapy regimens consisted of methotrexate, vinblastine, doxorubicin, and cisplatin. Sixteen (7.1%) of the 226 patients received neoadjuvant chemotherapy, whereas 60 (26.5%) patients underwent adjuvant chemotherapy and 21 (9.3%) underwent adjuvant radiation therapy for positive lymph node status and/or extravesical extension. Phenotypically normal urothelium was obtained from nine spinal cord injury and/or neurogenic bladder patients after simple cystectomy.

Tissue microarray. The index tumor was defined as the largest and/or highest tumor stage and grade. Tissue microarray blocks were constructed by taking core samples from morphologically representative areas of paraffin-embedded tumor tissues and assembling them on a recipient paraffin block. This was done with a precision instrument (Beecher Instruments, Silver Spring, MD) that uses two separate core needles for punching the donor and recipient blocks and a micrometer-precise coordinate system for assembling tissue samples on a block. For each case, three replicate 0.6-mm core diameter samples were collected and placed on separate recipient blocks. All samples were spaced 0.5 mm apart. Five-micrometer sections were obtained from the microarray and stained with H&E to confirm the presence of tumor and to assess the tumor histology. Tumor samples were randomly arranged on the blocks. Tissue array readings were confirmed by a pathologist with experience in immunohistochemical techniques and interpretation (R.A.).

Sample tracking was based on coordinate positions for each tissue spot in the tissue microarray block; the spots were transferred onto tissue microarray slides for staining. This sample tracking system was linked to a Microsoft Excel database containing demographic, clinical, pathologic, and survival data on each patient.

Immunohistochemistry and scoring. We did Ki-67 immunohistochemical staining using serial sections from the paraffin-embedded tissue microarray blocks. We used bright-field microscopy imaging coupled with advanced color detection software (Automated Cellular Imaging System, ChromaVision Medical Systems, Inc., San Juan Capistrano, CA) to detect, classify, and count stained cellular objects based on predetermined color morphology. The array was read according to the given tissue microarray map, each core was scored individually, and the results were presented as the mean of the three replicate core samples. Multiple known positive control sections were included in each run. Tumor sections with the primary antibodies substituted with rabbit immunoglobulin fraction and/or IgG monoclonal antibodies were used as negative controls. We obtained the mean, maximum, range, and SD of staining intensity and percentage positive nuclei/area measurements by using 10 random hotspots within each specimen. The mean of the triplicate cores was calculated for data analysis. Ki-67 immunoreactivity was considered altered when samples showed >20% nuclear reactivity. This definition was used according to the commonly used cutoff values ranging from 0% to 40% in TCC and other human cancers and also based on the examination of our staining data (4–6, 8, 13, 14). In a preliminary study, we assessed the discriminative value of Ki-67 as categorical variable with serial increments of cutoffs ranging from 5% to 90% positive cells with regard to bladder cancer prognosis (data not shown). Kaplan-Meier analyses revealed that the Ki-67cutoff of 20% was the best discriminator for both bladder cancer progression and survival (data not shown).

Statistical analysis. The Fisher's exact test and the χ2 test were used to evaluate the association between molecular markers and clinicopathologic variables. Differences in variables with a continuous distribution across dichotomous or ranked categories were assessed using the Mann-Whitney U test or the Kruskal-Wallis nonparametric ANOVA, respectively. The Kaplan-Meier method was used to calculate survival functions, and differences were assessed with the log-rank statistic. Univariate and multivariate survival analyses were done using the Cox proportional hazard regression model. Statistical significance in this study was set as P ≤ 0.050. All reported P values were two sided. All analyses were done with SPSS (version 13.0).

Results

Median age at radical cystectomy was 66.2 years (range, 34.2-88.0 years). Median number of lymph nodes removed was 17 (range, 10-53). Median time from last TUR to radical cystectomy was 1.7 months (range, 0.1-11.3 months). There were no differences in age at time of cystectomy, time from last TUR to radical cystectomy, and number of lymph nodes removed at radical cystectomy between normal and altered Ki-67 status (Ps ≥ 0.409).

Normal bladder urothelium from nine control patients showed no expression of Ki-67. Altered expression of Ki-67 was observed in 96 of 226 (42.5%) primary TCC from cystectomy specimens. Ki-67 was expressed in 28 of 50 (56%) of the radical cystectomy specimens and 27 of 50 (54%) of the matched lymph node specimens. The concordance rate of Ki-67 expression between matched cystectomy and malignant lymph node specimens was 82% with only 9 of 50 (18%) discordant cases.

Association of Ki-67 expression with molecular and pathologic characteristics of 226 consecutive patients treated with radical cystectomy for TCC of the urinary bladder

Disease recurred in 101 of 226 (44.7%) patients and 100 of 226 (44.2%) patients were dead at the time of analysis. Of these 100 patients, 82 (82%) patients died of metastatic bladder cancer, and 18 (18%) patients died of other causes without evidence of disease progression. The mean follow-up was 51.0 months (median, 36.9 months; range, 1.3-183.4 months) for those patients alive at the time of analysis. Kaplan-Meier analyses revealed that Ki-67 overexpression was significantly associated with an increased probability of disease recurrence (Fig. 1
) and bladder cancer-specific mortality (Fig. 2
).

Multivariable Cox regression analysis of pathologic features for the prediction of disease recurrence and disease-specific mortality in 210 consecutive patients treated with radical cystectomy for TCC of the urinary bladder

Multivariable Cox regression analysis of pathologic features for the prediction of disease recurrence and disease-specific mortality in 91 consecutive patients treated with radical cystectomy for organ-confined TCC of the urinary bladder after excluding patients who received neoadjuvant or adjuvant chemotherapy

Discussion

Approximately 50% to 60% of patients diagnosed with muscle-invasive TCC of the urinary bladder will develop metastatic progression after local therapy with curative intent, resulting in ∼12,000 deaths annually (1, 2). Although pathologic staging after local therapy is the most important prognosticator, its value for predicting clinical outcomes remains limited. A reliable predictor of metastatic progression would enhance our ability to identify patients who would benefit from adjuvant therapy and spare those who would not the toxicity associated with adjuvant therapies.

Several biomarkers, including proliferation-associated molecule Ki-67, have shown promise in their ability to stratify patients according to their risk for disease progression (15). Ki-67 is an established marker of cell proliferation, present during the G1, S, G2, and M stages of the cell cycle. In addition to Ki-67 antibody, MIB-1 antibody also detects the Ki-67 antigen but can be used on formalin-fixed, paraffin-embedded tissues. Staining for Ki-67 with MIB-1 is a simple and reproducible technique for assessing cell proliferation in bladder carcinoma and can be done on a small amount of tissue taken (3). Ki-67 expression is independently associated with clinical outcome after local therapy in several tumor types, such as breast, soft tissue, lung, cervix, melanoma, hepatocellular carcinoma, and prostate (4–6, 13).

Recently, investigators have established Ki-67 expression as an independent predictor of disease recurrence, progression, and response to intravesical therapy in patients with nonmuscle-invasive bladder cancer (7–10). In contrast, only few studies have examined the importance of Ki-67 expression in patients with muscle-invasive, locally advanced, or metastatic bladder cancer. Popov et al. (11), in a heterogeneous cohort of 114 patients treated with TUR or radical cystectomy, concluded that Ki-67 expression was independently associated with disease recurrence. Unfortunately, subgroup analysis of Ki-67 expression in patients with muscle-invasive and advanced TCC was not done. In a cohort of 75 patients treated with radical cystectomy, Suwa et al. (12) found that Ki-67 expression was an independent prognosticator of patient survival. However, most patients in that series had locally advanced or node-positive disease. Frank et al. (14) examined the expression of Ki-67 in the lymph node metastases from 139 patients who underwent cystectomy for TCC at their institution and found no association between Ki-67 and disease-related outcomes. However, when the analysis was limited to patients who were treated with adjuvant chemotherapy (n = 37 patients), there was a significant association between Ki-67 expression and distant metastases (P = 0.049).

We have shown, in one of the largest cohorts of patients with advanced TCC examined to date, that Ki-67 overexpression was significantly associated with advanced pathologic stage, higher tumor grade, lymphovascular invasion, and metastases to lymph nodes. Most importantly, Ki-67 expression was independently associated with both disease recurrence and bladder cancer-specific mortality when evaluated in the subgroup of patients with organ-confined disease (<pT3 N0). Patients with lymph node–positive disease are usually recommended to undergo adjuvant chemotherapy but it is not clear which patients with organ-confined disease will benefit from adjuvant therapies. As such identifying factors that may predict a worse outcome will improve our ability to counsel such patients about the best management strategy.

Several limitations of this investigation should be noted. First and foremost are the limitations inherent to the reliability and reproducibility of immunohistochemical techniques. Immunohistochemistry is semiquantitative and highly dependent on a range of poorly controlled variables, including antibody concentration, choice of antibody, variability in the interpretation and stratification criteria, and inconsistency in specimen handling and technical procedures. To reduce the number of variables in immunohistochemistry analysis, we have chosen to use tissue microarrays and an automated autostainer. This approach eliminates differential antigen retrieval and staining conditions as possible variables. Another limitation of immunohistochemical staining is the variability in the commonly used visual scoring system. These scoring methods are subjective and are subject to human variability. In the current study, we used a reproducible and accurate standardized, automated scoring system for assessing biomarker expression in tissue sections based on bright-field microscopy imaging coupled with advanced color detection software (16–18). Another limitation is the small sample size and relatively short follow-up, which may have limited our ability to detect an association between Ki-67 expression and bladder cancer-specific survival when evaluated in all patients.

Conclusions

Ki-67 immunostaining adds significant and independent prognostic information in patients treated with radical cystectomy for bladder TCC. Ki-67 expression may help identify patients with pathologically organ-confined bladder TCC who are at increased risk for disease progression and may benefit from adjuvant therapies. The clinical usefulness of this marker prompts large, prospective, multi-institutional comparative studies using standardized immunohistochemical assessment techniques.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.